RNA modification and the regulation of partial EMT in head and neck cancer

项目总结 头颈部鳞状细胞癌(HNSCC)是癌症相关死亡的第六大原因， 大多数死亡可归因于肿瘤转移和治疗失败。因为大多数HNSCC病例 由于吸烟和酒精暴露，这些肿瘤是高度异质性的，非常复杂 诊断、治疗和对这种疾病的生物学的调查。我们最近进行了单细胞核糖核酸- 对HNSCC中的单链RNA序列(scRNA-seq)进行测序，并鉴定出部分上皮间充质(p-EMT)转录 预测不良临床结果的计划，包括淋巴结转移和存活率降低(PURAM 等人，细胞)。了解控制HNSCC p-EMT计划的调控因素是至关重要的 在p-EMT等复杂途径中以多个基因为靶点的重要性尤其具有挑战性，但 显著改善HNSCC结局和治疗决策的潜力。RNA修饰 蛋白质可以直接读写RNA上的核苷酸标记，已经成为这类令人兴奋的一类 调节蛋白。因为这些蛋白质调节多个RNA转录本，靶向RNA修饰 蛋白质可能会扰乱多个疾病相关基因的表达，从而降低出现耐药性的可能性。在……里面 HNSCC，我们发现了一种催化N6-甲基腺苷的RNA甲基转移酶METTL3 (M6A)修饰，是HNSCC p-EMT计划的关键调节因子。在初步研究中，我们有 发现HNSCC中METTL3的扰动破坏了p-EMT信号并减少了体外侵袭。 然而，METTL3和其他RNA修饰蛋白发挥作用的确切机制 人们对此知之甚少，但可能取决于转录本稳定性和/或翻译的变化。因此，一个更好的 了解METTL3如何调节HNSCC中的p-EMT可能会改善合理的药物设计和 未来基于小分子和生物的筛选寻找有效的表位转录 治疗学。我们假设METTL3通过干扰HNSCC中p-EMT的翻译而拮抗p-EMT。 关键的p-EMT靶基因。要测试METTL3中的哪些域是其功能所必需的，我们将首先执行 HNSCC细胞系和患者来源的异种移植有机物(PDXO)的复杂结构-功能分析 (目标1)。我们将使用最先进的质谱学来确定m6A功能所需的结构域 方法，同时也映射关键结构域的结合伙伴。要确定METTL3是否控制 P-EMT基因转录或翻译(或两者兼而有之)，我们将利用先进的生化技术，包括 MERIP-seq研究p-EMT RNA转录本的稳定性和多聚体图谱及PAR-CLIP的研究 METTL3对p-EMT基因翻译的影响(目标2)。这些研究将提供不可或缺的洞察力 探讨METTL3指导HNSCC肿瘤发生的机制，特别是其对 P-EMT信号转导，从而揭示METTL3结构域，这些结构域可能是新疗法的靶点 有效且有针对性地治疗HNSCC。

PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer-related mortality, with the majority of deaths attributable to tumor metastasis and failures in treatment. Because most cases of HNSCC result from tobacco and alcohol exposure, these tumors are highly heterogeneous, greatly complicating diagnosis, treatment, and investigations into the biology of this disease. We recently performed single cell RNA- sequencing (scRNA-seq) in HNSCC and identified a partial epithelial-to-mesenchymal (p-EMT) transcriptional program that is predictive of poor clinical outcomes including nodal metastasis and diminished survival (Puram et al., Cell). Understanding the regulatory factors that control the p-EMT program in HNSCC is of critical importance as targeting multiple genes in a complex pathway such as p-EMT is particularly challenging, yet has the potential to significantly improve HNSCC outcomes and treatment decision-making. RNA modification proteins, which can directly read/write nucleotide marks on RNA, have emerged as one exciting class of such regulatory proteins. Because these proteins modulate multiple RNA transcripts, targeting RNA modification proteins may disrupt expression of multiple disease-related genes and make resistance less likely to emerge. In HNSCC, we have found that METTL3, an RNA methyltransferase which catalyzes the N6-methyladenosine (m6A) modification, is a key regulator of the p-EMT program in HNSCC. In preliminary studies, we have discovered that perturbation of METTL3 in HNSCC disrupts p-EMT signaling and reduces invasion in vitro. However, the precise mechanisms by which METTL3 and other RNA modification proteins exert their function are poorly understood, but may depend on changes in transcript stability and/or translation. Thus, a better understanding of how METTL3 modulates p-EMT in HNSCC is likely to improve rational drug design and future small molecule- and biologically-based screens in search of effective epitranscriptomic therapeutics. We hypothesize that METTL3 antagonizes p-EMT in HNSCC by disrupting the translation of critical p-EMT target genes. To test which domains in METTL3 are essential for its function, we will first perform sophisticated structure-function analyses in HNSCC cell lines and patient-derived xenograft organoid (PDXOs) (Aim 1). We will determine which domains are required for m6A function using state-of-the-art mass spectrometry methods, while also mapping the binding partners of the critical domains. To determine if METTL3 controls the transcription or translation (or both) of p-EMT genes, we will utilize advanced biochemical techniques including meRIP-seq to study the stability of p-EMT RNA transcripts and polysome profiling and PAR-CLIP to investigate the effects of METTL3 on translation of p-EMT genes (Aim 2). These studies will provide indispensable insight into the mechanism by which METTL3 directs HNSCC tumorigenesis, specifically focusing on its regulation of p-EMT signaling, and thereby revealing METTL3 domains that could be targeted by new therapeutics to more effectively and specifically treat HNSCC.